The majority of potential donor lungs are rejected for transplantation and thus many patients die while waiting for a suitable donor. Donor lungs are typically retrieved from individuals after brain death; however, there is renewed interest in utilizing non-heart- beating donor (NHBD) lungs retrieved after cardiac death. The major challenge for utilization of NHBD lungs is the difficulty in assessing function after a warm ischemic period which results in significant injury. Two novel approaches show promise to greatly expand the utilization of NHBD lungs: 1) use of a lung box for ex vivo lung perfusion (EVLP) to assess and rehabilitate lungs prior to transplantation, and 2) use of adenosine 2A receptor (A2AR) agonist to rehabilitate lungs and prevent ischemia-reperfusion (IR) injury. This proposal combines these two approaches to perfect a lung box-based method of EVLP to assess and therapeutically rehabilitate NHBD lungs for successful transplantation. We have demonstrated the rehabilitation and successful transplantation of NHBD porcine lungs after EVLP with an A2AR agonist, and we have demonstrated that an adenosine 2B receptor (A2BR) antagonist attenuates IR injury. Thus our proposed studies will test the hypothesis that NHBD lungs can be rehabilitated for successful transplantation by EVLP-mediated therapy using an A2AR agonist or A2BR antagonist. We will test this hypothesis with three aims. Aim 1 will use a porcine lung transplant model to determine if EVLP with A2AR agonist can safely extend both warm and cold ischemic times of NHBD lungs. Aim 2 will determine if EVLP with A2AR agonist therapy will rehabilitate marginal and previously rejected human donor lungs to an acceptable state for transplantation. Aim 3 will determine if EVLP with A2BR antagonist will rehabilitate porcine and human NHBD lungs. Within each aim, we will also determine if the rehabilitation of NHBD lungs by EVLP entails the inhibition of NKT cell activation, epithelial cell activation and neutrophil infiltration. This project addresses two major problems that plague lung transplantation: far too few suitable lungs for transplant and primary graft failure after transplat due to IR injury. If NHBD lungs can be safely rehabilitated for transplantation, then the lung donor shortage could be eliminated. Thus this project will provide a means to greatly extend the donor lung pool size and support clinical trials aimed at saving many more lives of those patients on the lung transplant waiting list.